Estrogen Regulation and Localization of Galanin Gene Expression in ...

BIOLOGY OF REPRODUCTION 49, 1245-1250 (1993)

Estrogen Regulation and Localization of Galanin Gene Expression in the Rat Uterus' MARIA VRONTAKIS, 2 INGO SCHROEDTER, VALERIANO LEITE, and HENRY G. FRIESEN Departmentof Physiology, University of Manitoba, Winnipeg, Manitoba, CanadaR3E OW3 ABSTRACT To determine whether galanin is a target for estrogenic regulation in the rat uterus, we measured the effects of estrogen on galanin mRNA expression in the uterus of ovariectomized rats and compared the results with the regulation of galanin in the anterior pituitary. Treatment of the animals with a single dose of 17[-estradiol resulted in a rapid transient increase of galanin mRNA, similar to that in the pituitary of the same animals, with a peak at 3 h after stimulation and a return to prestimulation levels after 24 h. No galanin mRNA was detected in ovariectomized control animals in either tissue. By in situ hybridization of rat uterus 3 h after estrogen stimulation, we found that galanin mRNA was localized in the endometrium in stromal cells that are close to the lumen. There was no hybridization in the myometrium of the estrogen-treated animals. Surprisingly, and strikingly different from results in the pituitary, in the uterus with a constant and prolonged exposure to estrogen by diethylstilbestrol (DES) implants, the induction of galanin mRNA was only transient, with return to baseline levels by 24 h despite the continued presence of DES. On the other hand, in the pituitary there was a rapid and sustained increase of galanin mRNA. These data demonstrate that 1) one of the initial steps in the mechanism of estrogen stimulation in the rat uterus involves galanin gene expression; 2) in the uterus galanin mRNA is expressed in stromal cells of the endometrium; and 3) the regulation of galanin mRNA expression by estrogen in the pituitary and the uterus is markedly different.

INTRODUCTION A major effect of estrogen in the uterus is stimulation of DNA synthesis and cell proliferation. The uterine response initially involves hyperaemia, increased wet weight, glucose metabolism, histamine depletion, and RNA polymerase activity followed by hypertrophy and hyperplasia within 24 h of hormone treatment [1-4]. One of the earliest events that is likely to be important in the proliferative response of the uterus to estrogen is the activation of proto-oncogene transcription (c-myc, c-fos, etc.), which encode nuclear regulatory proteins [5-7]. The late-phase responses [48 h] include an increase in protein synthesis and an increase in uterine dry weight and DNA contents [8-9]. It has been postulated that estrogen controls cell proliferation, acting indirectly by the local paracrine, autocrine, or systemic (endocrine) production of growth factors, or directly, as a mitogen for target cells. The leading general hypothesis for the mechanism of hormone action is that the estrogen receptor complex binds to cs-acting enhancer-like responsive elements located within or near responsive genes to influence promoter activity. Galanin is a newly isolated neuropeptide widely distributed throughout the central nervous system of the rat [10]. In the anterior pituitary, estrogen dramatically elevates galanin mRNA [11-12]. In contrast, galanin levels in the hypothalamus and neurointermediate lobe are not altered by estrogen [13]. To determine whether galanin is a target for Accepted July 15, 1993. Received March 16, 1993. 'This study was supported by the National Cancer Institute and the Medical Research Council of Canada. V. Leite is a recipient of a fellowship from Junta Nacional de Investigacao Cientifica e Tecnol6gica, Portugal (BD/845/90-ID). 2Correspondence: M.E. Vrontakis, M.D., Department of Physiology, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E OW3. FAX: (204) 774-9517.

estrogen regulation in the rat uterus, we measured the effects of estrogen on galanin gene expression in that organ. MATERIALS AND METHODS Animals Female ovariectomized Fisher 344 rats weighing 150 g (Charles River Breeding Labs., Canada) were housed under central lighting (lights-on, 0600-1800 h) and temperature (22°C). Free access to laboratory chow and tap water was provided. All of the experimental procedures were performed in accordance with protocols authorized by the University of Manitoba Commmittee on Animal Care, which conform to the procedures approved by the Canadian Council on Animal Care. Ovariectomy was performed at least 21 days prior to the experiment. In the first set of experiments, different groups of rats received a single injection of 171-estradiol (10 g/100 g BW) or vehicle (10% ethanol:90% saline) and were killed by decapitation at different time points as indicated. In the second set of experiments, different groups of rats received an implant (10 mg) of diethylstilbestrol (DES) for 3 h, 2 days, and 7 wk. In the dose-response experiment we treated rats with different doses of 1703-estradiol ranging from 0.75 jLg/kg to 10 jig/kg. Four animals per group were used at each time point. Each experiment was performed at least three times. In each animal the pituitary and the uterus were quickly dissected and immediately frozen on dry ice. RNA Extraction and Northern Blot Analysis

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RNA was prepared from frozen tissue by the guanidium isothiocyanate/cesium chloride method [14]. Total RNA (20 pig) was electrophoresed on a denaturing agarose formaldehyde gel, and the RNA was transferred to a Nitroplus 2000

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probe and the B-mercaptoethanol for 2-4 h. The slides were then dehydrated in 100% ethanol and air-dried. The probe used was a copy of galanin RNA labeled with [35S]uridine 5'-triphosphate to a specific activity of 2-4 x 105 dpm/ng. We used the Promega Riboprobe system (Promega Biotec, Madison, WI) with Pharmacia (Piscataway, NJ) polymerases. The hybridization mix contained 50% formamide, 0.3 M NaCl, 20 mM Tris HC1 (pH 8.0), 1 mM EDTA, single-strength Denhardt's solution (0.02% each BSA, Ficoll 200, polyvinylpyrrolidone), 500 pig/ml salmon testes DNA, 50 ,Lg/ml polyadenylic acid, 200 mM P3-mercaptoethanol. The probe was added at a concentration of 0.1 ng/pl, usually 20 1L/ section under a 22-mm siliconized coverslip. The slides were set up hot -60°C on a slide warmer and then submerged into 50°C mineral oil. The slides were incubated for 10-18 h. The stringent final washes were 67°C with 50% formamide, 0.3 M NaCl, 20 mM Tris, 1 mM EDTA, and 100 mM P-mercaptoethanol for 30 min and 0.1% SSC at 67 0C for 15 min. The tissues were treated with RNAses "Ti" and "A." The slides were dehydrated with 50, 70, and 90% ethanol containing 0.3 M ammonium acetate and then dipped in Kodak NTB2 emulsion diluted 1:1 with water, dried, and exposed at 4°C desiccated. Exposed slides were processed with D-19 Kodak Developer and Kodak fixer. The tissues were counterstained with Harris hematoxylin and eosin. FIG. 1. Time course of galanin mRNA induction by estrogen in rat uterus. Total RNA (20 p.g) isolated from rat uteri were used in this Northern blot analysis with rat galanin cDNA probe. Lane 1: control group. Lanes 26: group of animals decapitated at different intervals after a single injection of estradiol-17P (10 ig/100 g BW). Lane 2 = 1 h. Lane 3 = 3 h. Lane 4 = 6 h. Lane 5 = 12 h. Lane 6 = 24 h. The positions of 18S and 28S ribosomal RNA are indicated, and the variability of RNA loading and transfer is shown in the lower part of the figure (the 28S ribosomal cDNA probe was used to reprobe the blot).

membrane by blotting in 20-strength saline sodium citrate (SSC). The equivalent loading and transfer of RNA was verified by use of 28S cDNA probe to reprobe the blots. Hybridization was performed at 42°C in 50% formamide. Final washing conditions were 0.1-strength SSC, 0.1% SDS at 65°C for 30 min. Autoradiography was done using X-Omat AR film (Eastman Kodak, Rochester, NY) and a lighting Plusk enhancing screen at -70 0C for 1-7 days. In Situ Hybridization Tissues were processed to wax and blocked in Ameraffin (CanLab). The blocks were cut at 5 ptm, floated onto aminoalkysilane-treated slides [15], and placed in a 50°C oven for 12-18 h. In situ hybridization was performed according to the protocol of Angerer et al. [16] with the following modifications. Briefly, the tissue was permeabilized with 0.2 N HC1 30 min at 20-24°C and then treated with proteinase K (1 Fpg/ml) at 37°C for 15 min. The slides were acetylated and placed in a 50°C prehybridization solution that contained all the reagents of the hybridization mix minus the

Dot-Blot Hybridization The dose response of different amounts of galanin RNA to estrogen was determined by dot-blot hybridization as described by White and Bancroft [17]. RESULTS Early Increase of Galanin Expression by Estrogen in the Rat Uterus The level of galanin mRNA was measured in uterine RNA isolated from ovariectomized rats killed at different time points after single injection of 171-estradiol. Analysis was performed by Northern blot hybridization using the 690bp cDNA clone of the rat preprogalanin [11]. Figure 1 shows the results of one representative experiment as an autoradiograph of the nitrocellulose filter. For estimating the equivalent loading and transfer, the same blot was hybridized with a cDNA probe of the rat 28S ribosomal RNA. The galanin mRNA is not detectable in the uteri of untreated ovariectomized animals (Fig. 1, lane 1), whereas a single injection of estrogen (10 pLg/100 g BW) resulted in a rapid transient increase of galanin mRNA with a peak at 3 h (Fig. 1, lanes 2-6), similar to that we have previously shown in the rat pituitary after estrogen treatment [11]. The size of the transcript in the uterus was the same, 0.9 kb, as previously reported in the rat pituitary. Since one of the initial uterine responses to the estrogenic stimulus is the rapid induction of certain oncogenes, we compared the induc-

GALANIN IN UTERUS

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FIG. 2. Comparison of the effect of estrogen in the induction of c-myc and galanin mRNA expression in the rat uterus. Total RNA from control and estrogen-treated uteri were used in this Northern blot. The same blot was hybridized to c-myc and rat galanin cDNA probes sequentially.

tion of galanin with that of c-myc in the uterus. Figure 2 shows the same blot hybridized with a c-myc cDNA probe (an 8.3-kb fragment encoding exons 1, 2, and 3 [18] and galanin cDNA probe. As shown in Figure 2, the pattern of induction of the two genes is very similar for the first 12 h. Dose-Related Increase in Galanin mRNA Expression by Estrogen in the Rat Uterus We examined the increase in uterine galanin mRNA expression as a function of the dose of estrogen. Groups of rats received injections of increasing doses of estradiol173 (0.75-10 ptg/kg) and were killed 3 h after injection. Figure 3 shows the levels of galanin mRNA obtained by use of dot blot hybridization of different amounts of RNA (102.5 pg total RNA) isolated from rat uterus of animals treated with increasing doses of estrogen. There is clearly an increase in the levels of the galanin mRNA that is related to the dose of estrogen and the amount of RNA encoded, indicating a positive dose response of the galanin mRNA level to estrogenic stimulation. Effects of Chronic Estrogen Treatment In previous experiments, we demonstrated that constant and prolonged exposure of rats to estrogen (2 days-7 wk) by administration of DES implants (10 mg) resulted in an induction of the galanin transcript in the anterior pituitary within 3 h, which continued to be highly expressed throughout the 7 wk. In the present study, however, we found that in the uterus of the same animals the induction of the galanin transcript after prolonged exposure to estro-

gen was transient, with return to baseline levels by 24 h despite the continued presence of estrogen. This is strikingly different from the pattern found in the pituitary. The same blot was probed with 13-actin (another estrogen-regulated gene) and the presence of the [3-actin transcript was identified in all lanes. Figure 4 is a representative Northern blot of rat pituitaries and uterus after chronic DES treat-

FIG. 3. Dose response of galanin mRNA expression to estrogen in the rat uterus. Autoradiogram of a dot blot of total uterine RNA from rats treated with 0.75, 2.5, 5, and 10 tIg/kg, 17p-estradiol (E2).The amount of RNA loaded per well is indicated.

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FIG. 4. Effect of chronic estrogen treatment on the expression of rat galanin mRNA in the pituitary and uterus. Total RNA (20 Lg)from the pituitary (lanes 1-4) and uterus (lanes 5-8) of the same animals was used in Northern blot hybridized with the rat galanin cDNA probe. Lanes 1 and 5, control group; lanes 2 and 6, animals implanted with DES for 3 h; lanes 3 and 7, animals implanted with DES for 48 h; lanes 4 and 8, animals implanted with DES for 7 wk.

ment. The same results were obtained by using estradiol1713 implants (data not shown). Comparison of the induction of the galanin mRNA levels at 3 h after estrogen stimulation reveals that in the uterus the induction was 70% of the induction in the pituitary at the same point under exactly the same experimental conditions (control = 100, pituitaries = 1025 + 120, uteri = 750 + 84). In Situ Hybridization To determine the cellular site of galanin gene expression in the uterus, we used in situ hybridization. The results of hybridization of longitudinal uterine sections 3 h after estrogen administration are shown in Figure 5. No hybridization was detected with sense riboprobe (Fig. 5a), whereas hybridization was detected with the antisense riboprobe (Fig. 5, b-d). Galanin was localized in the endometrium, with very dense grains in stromal cells that are very close to the lumen (Fig. 5d). There was no hybridization in the myometrium of the estrogen-treated animals. DISCUSSION We investigated the expression of the the rat uterus after estrogen treatment. pressed in the ovariectomized rat uterus estrogen treatment with a peak at 3 h, but

galanin gene in Galanin is exvery early after this induction is

transient, returning to prestimulation levels within 24 h. Recent studies have demonstrated that in the uterus, estrogens modulate the expression of genes encoding proto-oncogenes [5, 7, 19, 20] growth factors [21, 22], and growth factor receptors [23]. Since proto-oncogenes, growth factors, and growth factor receptors clearly are associated with the regulation of cell division, it has been suggested that the activation of proto-oncogenes and/or the production of growth factors and their receptors may mediate the uterine growth response induced by estrogens [24]. The time course of estrogen-stimulated expression of galanin in the uterus is very similar (as is shown in Fig. 2) to that of c-myc and coincides with the time course established for the development of many early uterine growth responses. The estrogen responsiveness of various markers in the uterus can be a direct effect of occupied receptor on transcription of specific genes or an indirect effect mediated by inducible intermediates. However, previous work by Kaplan et al. [25] failed to detect an ERE consensus sequence in the 5' flanking region of the rat galanin gene, although this finding does not exclude the possibility of an ERE sequence different from the consensus sequence described so far or an ERE sequence located far away from the promoter of the galanin gene. The dose response of galanin to estrogen indicates that it is quite sensitive to estrogen, with response as low as 0.750 pg/kg of 173-estradiol. Surprisingly, after chronic estrogen treatment the induction of galanin mRNA in the uterus is transient despite the continued presence of estrogen. On the other hand, in the pituitary of the same animals there is a rapid and sustained increase of galanin mRNA levels. The increase of galanin expression in the pituitary after estrogen treatment could be partially attributed to the increase in the number of lactotrophs since galanin colocalizes in lactotrophs [26, 27], although the increase of galanin gene expression occurs even before any hyperplasia of lactotroph take place (24-48 h after estrogen treatment). This difference in the expression of galanin in the uterus and pituitary existed whether we used 1713-estradiol or DES (data not shown). The reason for this tissue difference in the response of galanin to estrogen is not clear. It is obvious from Figure 4 that another estrogen-responsive gene (-actin) continued to be expressed after prolonged exposure to estrogen. On the other hand c-fos and cun in the presence of continuous estrogenic stimulus become refractory after an initial increase in transcription [6, 7], similar to the pattern of galanin in the uterus. Recently Csikos et al. [28] described a similar situation for c-fos in the anterior pituitary of estrogen-stimulated rats, where estrogen induced both c-fos and galanin gene expression. Prolonged exposure to estrogen, however, appears to depress c-fos expression, whereas galanin gene expression continues to rise, indicating a transient expression of the c-fos gene in the anterior pituitary. Csikos et al. [28] suggested that c-fos may participate in regulating galanin expression since an AP-1 site is present in the up-

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stream region of the galanin gene. Transient accumulations of specific mRNAs suggests that a mechanism must exist to counterbalance the stimulatory effect of estrogen on the activities of these promoters in uterine cells. On the other hand, instability or degradation of galanin mRNA in the rat uterus could account for the phenomena. At the moment we do not know exactly what mechanisms control the induction of the transient expression in the two tissues. The only knowledge that we have so far is that estrogen increases the transcription of the galanin gene in the anterior pituitary by almost 60-fold [25]. By in situ hybridization, galanin was localized in the endometrium, in stromal cells that are close to the lumen. This finding is consistent with our finding [29] that galanin is expressed in decidual tissue during pregnancy i'n the rat, since decidual cells are considered to arise by in situ differentiation of stromal fibroblast type cells. The localization of galanin, particularly in stromal cells, indicates that it might act through an autocrine or paracrine effect to mediate the effect of estrogen in the uterus. There is now compelling evidence that stromal-epithelial cell interaction is necessary for the normal response of the uterus to estrogen [30]. Stromal tissue may provide, in addition to mechanical support, growth factors for epithelial cells that mediate estrogen responsiveness. Further studies are necessary to establish the exact mechanism involved in the regulation of galanin in these two tissues. ACKNOWLEDGMENT The secretarial assistance of Ms. Judy Olfert is very much appreciated.

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9. Oamby VE, Korach KS. The influence of 17-8 estradiol on pattern of cell division of the uterus. Endocrinology 1984; 114:694-699. 10. Michener SR, Aimare LD, Yakshi TL, Go VLW. Distribution of galanin like immunoreactivity in the pig, rat and human central nervous system. Peptides 1990; 11:1217-1223. 11. Vrontakis ME, Peden LM, Duckworth ML, Friesen HG. Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary human mRNA. J Biol Chem 1987; 262:16755-16760. 12. Kaplan LM, Gabriel SM, Koenig JI, Sunday ME, Spindel ER, Martin JB, Chin WW. Galanin is an estrogen-inducible secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 1988; 85:7408-7412. 13. Vrontakis ME, Yamamoto T, Schroedter IC, Nagy JI, Friesen HG. Estrogen induction of galanin synthesis in the rat anterior pituitary gland demonstrated by in situ hybridization and immunohistochemistry. Neurosci Lett 1989; 100:59-65. 14. Chirgwin JM, Przbyla AE, McDonald RJ,Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 1979; 18:5294-5299. 15. Henderson C. Aminoalkylsilane: an inexpensive, simple preparation for slide adhesion. J Histochem 1989; 12:123-124. 16. Angerer LM, Stoler MH, Angerer RC. In situ hybridization with RNA probes: an annotated recipe. In Valentino KL, Eberwine JH, Barches JD (eds.), In Situ Hybridization: Application to Neurobiology. New York: Oxford University Press; 1987: 43-70. 17. White BA, Bancroft FC. Cytoplasmic dot hybridization simple analysis of relative mRNA levels in multiple small cell or tissue samples. J Biol Chem 1982; 257:8569 8577. 18. Dalla Favera R, Gelmann EP, Martinnoti S, Franchini G, Papas T, Gallo RC, WongSteel J. Cloning and characterization of different sequences related to the oncogene (v-myc) of aviary myelocytomutosis virus. Proc Natl Acad Sci USA 1982; 79:6497-6503. 19. Murphy LJ,Murphy LC, Friesen HG. Estrogen interaction of N-myc and c-myc proto-oncogenes expression in the rat uterus. Endocrinology 1982; 120:18821888. 20. Travers MT, Knowles JT. Oestrogen induced expression of oncogenes in the immature rat uterus. FEBS Lett 1987; 211:27-30. 21. DiAugustine RP, Petrusz P, Bell G, Brown CF, Korach KS, McLachlan JA, Teng CT. Influence of estrogens on mouse uterine epidermal growth factor precursor protein and messenger RNA Endocrinology 1988; 122:2355-2363. 22. Huet-Hudson YM, Chakraborty C, De SK, Suyuki Y, Andrews GK, Dey SK. Estrogen regulates the synthesis of epidermal growth factor in uterine epithelial cells. Mol Endocrinol 1990; 4:510-523. 23. Mukku VR, Stancel GM. Regulation of epidermal growth factor receptor by estrogen. J Biol Chem 1985; 260:9820-9824. 24. Roves C, Spelsberg TC. Ovarian steroid action on gene expression: mechanism and models. Annu Rev Physiol 1989; 57:653-687. 25. Kaplan LM, Hooi S, Abraczinskas DR. Strauss RM, Davidson MB, Hsu DW, Koenig JI. Neuroendocrine regulation of galanin gene expression. In: Galanin. Wenner Gren Symposium Series 1992; Hkfelt Ed. Macmillan Press p 43. 26. Vrontakis ME, Neil JD. Inhibition of prolactin secretion by galanin antiserum. In: Program of the 7th annual meeting of the Endocrine Society; 1989; Seattle, WA. Abstract. 27. Steel JH, Gan G, O'Halloran DJ, Jones PM, Yanaihara N, Ishikawa H, Bloom SR, Polak JM. Galanin and VIP are colocalized with classical pituitary hormones and show plasticity of expression. Histochemistry 1990; 93:183-188. 28. Csikos T, McDonald JK, Koenig J. Estrogen induction of galanin and immediate early gene expression in the rat anterior pituitary gland. Abst Soc Neurosci 1992; 18:456.10. 29. Vrontakis ME, Schroedter IC, Cosby H, Friesen HG. Expression and secretion of galanin during pregnancy in the rat. Endocrinology 1992; 130:458-463. 30. Cooke PS, Uchiwa FDA, Fujii DK, Bern HA, Cunha GR Restoration of normal morphology and estrogen responsiveness in cultivated vaginal and unterine epithelia transplanted with stroma. Proc Natl Acad Sci USA 1986; 83:2109-2114.